U.S. patent application number 11/168017 was filed with the patent office on 2006-01-12 for compositions and methods for use against acne-induced inflammation and dermal matrix-degrading enzymes.
Invention is credited to Gary J. Fisher, Sewon Kang, John J. Voorhees.
Application Number | 20060009494 11/168017 |
Document ID | / |
Family ID | 27076996 |
Filed Date | 2006-01-12 |
United States Patent
Application |
20060009494 |
Kind Code |
A1 |
Voorhees; John J. ; et
al. |
January 12, 2006 |
Compositions and methods for use against acne-induced inflammation
and dermal matrix-degrading enzymes
Abstract
Acne-affected skin has been found to be accompanied by the
presence of matrix-degrading enzymes such as MMPs and neutrophil
elastase, induction of neutrophils, and a reduction in procollagen
biosynthesis. This invention treats scarring and inflammation
accompanying acne by administering, topically or systemically, at
least one of (i) an inhibitor of the matrix degrading enzymes and
(ii) a cytokine inhibitor that alleviates inflammation and thus
also alleviate neutrophil infiltration. Alleviating the matrix
degradation and renormalizing procollagen biosynthesis allows for
reduced inflammation and better natural repair of acne-affected
skin. Inhibiting cytokines alleviates induction of MMPs in resident
skin cells, and also alleviates inflammation with its concommitant
induction of neutrophils from the blood stream bringing MMPs and
elastase into the acne lesion. Dimishing the presence of
matrix-degrading enzymes in the acne lesion reduces imperfect
repair of the skin and thus decreases scarring in acne-affected
skin.
Inventors: |
Voorhees; John J.; (Ann
Arbor, MI) ; Kang; Sewon; (Ann Arbor, MI) ;
Fisher; Gary J.; (Ypsilanti, MI) |
Correspondence
Address: |
Bradley N. Ruben
Ste. 5A
463 First St.
Hoboken
NJ
07030
US
|
Family ID: |
27076996 |
Appl. No.: |
11/168017 |
Filed: |
June 27, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09852154 |
May 9, 2001 |
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11168017 |
Jun 27, 2005 |
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09576597 |
May 22, 2000 |
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09852154 |
May 9, 2001 |
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60134984 |
May 20, 1999 |
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Current U.S.
Class: |
514/340 |
Current CPC
Class: |
A61P 29/00 20180101;
A61K 31/07 20130101; A61P 17/10 20180101; A61P 39/06 20180101; A61K
8/22 20130101; A61K 31/41 20130101; A61K 8/671 20130101; A61K
31/4439 20130101; A61P 43/00 20180101; A61P 5/44 20180101; A61Q
19/00 20130101; A61K 2800/782 20130101; A61P 31/04 20180101 |
Class at
Publication: |
514/340 |
International
Class: |
A61K 31/4439 20060101
A61K031/4439 |
Claims
1. A composition for alleviating acne scarring, comprising a
compatible combination of: a non-retinoid inhibitor of a dermal
matrix-degrading enzyme; and an active ingredient selected from the
group consisting of comedolytics, antibacterials,
anti-inflammatories, retinoids, glucocorticoids, and compatible
mixtures thereof.
2. The composition of claim 1, wherein the inhibitor is an
inhibitor is selected from the group consisting of AP-1 inhibitors,
NF-.kappa.B inhibitors, elastase inhibitors, adhesion antagonists,
and mixtures thereof.
3. The compositon of claim 1, wherein the composition is applied
topically and is provided in combination with a
dermatologically-acceptable carrier.
4. The compositon of claim 1, wherein the composition is
administered systemically.
5. The composition of claim 1, wherein the active ingredient is a
retinoid.
6. The composition of claim 1, wherein the active ingredient is an
antibacterial.
7. The composition of claim 6, wherein the active ingredient is
benzoyl peroxide.
8. The composition of claim 1, comprising a combination of an MMP
inhibitor and a neutrophil elastase inhibitor.
9. The composition of claim 1, wherein the inhibitor is an
antioxidant.
10. A method for treating acne, comprising the steps of: orally
administering an active ingredient for the treatment of acne and
topically administering a non-retinoid, non-glucocorticoid
inhibitor of a dermal matrix degrading enzyme to acne-affected
skin.
11. The method of claim 10, wherein the active ingredient is a
retinoid or a tetracycline or derivative thereof.
12. The method of claim 10, wherein the inhibitor is selected from
the group consisting of AP-1 inhibitors, NF-.kappa.B inhibitors,
elastase inhibitors, selectin inhibitors, and compatible mixtures
thereof.
13. The method of claim 12, wherein the inhibitor of a dermal
matrix degrading enzyme is an MMP inhibitor.
14. The method of claim 10, wherein the inhibitor an
antioxidant.
15. The method of claim 10, wherein the inhibitor is applied
regularly from once every two days to twice daily.
16. The method of claim 15, wherein the inhibitor is applied
daily.
17. The method of claim 10, wherein the inhibitor comprises a
combination of an MMP inhibitor and an elastase inhibitor.
18. The method of claim 1, wherein the inhibitor is a direct MMP
inhibitor.
19. The method of claim 1, wherein the inhibitor is an indirect MMP
inhibitor.
20. A combined therapy for alleviating acne scarring, comprising a
compatible combination of: a non-retinoid inhibitor or antagonist
of an receptor sensitive to LPS-like material; and an active
ingredient selected from the group consisting of comedolytics,
antibacterials, anti-inflammatories, retinoids, glucocorticoids,
non-retinoid MMP inhibitors, and compatible mixtures thereof.
21. The combined therapy of claim 20, wherein the composition is
applied topically and is provided in combination with a
dermatologically-acceptable carrier.
22. The combined therapy of claim 20, wherein at least one of the
active ingredient and the inhibitor is administered topically.
23. The combined therapy of claim 20, wherein at least one of the
active ingedient and the inhibitor is administered orally.
24. The combined therapy of claim 20, where the combination is
provided as a single, topically applied composition.
25. The combined therapy of claim 20, wherein the inhibitor or
antagonist inhibits or antagonizes a TLR.
26. A method for treating acne, comprising the steps of:
administering an active ingredient for the treatment of acne and
administering a non-retinoid, non-glucocorticoid inhibitor or
antagonist of a receptor sensitive to LPS-like compounds induced or
produced by P. acnes.
27. The method of claim 26, wherein the active ingredient is a
retinoid or a tetracycline or derivative thereof.
28. The method of claim 26, wherein the inhibitor or antagonist is
administered topically and applied regularly from once every two
days to twice daily.
29. The method of claim 25, wherein the inhibitor or antagonist is
administered daily and the active ingredient is administered
topically.
30. The method of claim 26, wherein the inhibitor or antagonist
reduces the induction or production or signalling by
NF-.kappa..beta..
31. A method for treating acne, comprising administering a
non-retinoid inhibitor of NF-.kappa..beta. to a patient in need
thereof.
32. The method of claim 31, wherein the inhibitor affects the
production of NF-.kappa..beta. from TLRs.
33. A method for treating acne, comprising administering an
inhibitor that prevents CD-14 from activating toll-like
receptors.
34. The method of claim 33, wherein the CD-14 inhibitor is
administered topically.
Description
[0001] This application is a CIP of appln. Ser. No. 09/576,597,
which is based on appln. No. 60/134,984.
TECHNICAL FIELD
[0002] This invention involves protecting human skin from some of
the effects of acne, especially acne vulgaris, through the use of
the topically and/or systemically applied non-retinoid and
non-steriod compounds that diminish inflammation and
matrix-degrading enzymes in acne-affected skin.
BACKGROUND
[0003] Acne is a multifactorial disease, developing in the
sebaceous follicles. At least one agent thought responsible is the
anaerobe Propionibacterium acnes (P. acnes); in younger
individuals, practically no P. acnes is found in the follicles of
those without acne.
[0004] The disease of acne is characterized by a great variety of
clinical lesions. Although one type of lesion may be predominant
(typically the comedo), close observation usually reveals the
presence of several types of lesions (comedones, pustules, papules,
and/or nodules). The lesions can be either noninflammatory or, more
typically, inflammatory. In addition to lesions, patients may have,
as the result of lesions, scars of varying size. The fully
developed, open comedo (i.e., a plug of dried sebum in a skin pore)
is not usually the site of inflammatory changes, unless it is
traumatized by the patient. The developing microcomedo and the
closed comedo are the major sites for the development of
inflammatory lesions. Because the skin is always trying to repair
itself, sheaths of cells will grow out from the epidermis (forming
appendageal structures) in an attempt to encapsulate the
inflammatory reaction. This encapsulation is often incomplete and
further rupture of the lesion typically occurs, leading to
multichanneled tracts as can be seen in many acne scars.
[0005] In general, there are four major principles presently
governing the therapy of acne: (i) correction of the altered
pattern of follicular keratinization; (ii) decrease sebaceous gland
activity; (iii) decrease the follicular bacterial population
(especially P. acnes) and inhibit the production of extracellular
inflammatory products through the inhibition of these
microorganisms; and (iv) produce an anti-inflammatory effect. The
present treatments for acne following these principals typically
include: vitamin A acid (retinoic acid), known for its comedolytic
properties, administered topically (e.g., Retin-A.RTM. brand 0.025%
all-trans retinoic acid cream) or systemically (e.g., Accutane.RTM.
brand 13-cis retinoic acid); an antibiotic administered
systemically (e.g., tetracycline or one of its derivatives) or
topically (e.g., benzoyl peroxide, erythromycin, clindamycin,
azelaic acid); the use of other comedolytic agents such as
salicylic acid; or the use of systemic anti-androgens such as
cyproterone acetate and spironolactone (because androgens promote
sebum production, and sebum has been found to be comedogenic and
inflammatory), which may be administered in combination with an
estrogen. Atrophy, the most feared side effect of topical
glucocorticoids, is seen as an overall reduction in the dermal
volume and occurs as early as one week after superpotent-steroid
use. Systemic side effects of chronic glucocorticoid use include
suppression of the hypothalamic-pituitary-adrenal (HPA) axis,
Cushing's syndrome, glaucoma, and, in children, failure to thrive.
(Children, especially infants and young children, are at higher
risk for systemic side effects due to their greater surface-to-body
ratio. They also may not metabolize corticosteroids as well as
adults.) Withdrawal symptoms can appear after topical steriods have
been used for a long period of time. Severe flaring may occur when
isotretinoin (13-cis) therapy is started, and so concommitant use
of a steriod, and suboptimal doses of isotretinoin, are often
required at the start of therapy; additionally, retinoids generally
are teratogenic (inhibiting organogenesis as opposed to being
mutagenic).
[0006] The art has addressed inflammation and scarring caused by
acne as a secondary benefit to the treatment of the disease; that
is, if the acne is cured the factors causing scarring will be
eliminated. There is otherwise no treatment directed at preventing
scarring from acne. Neither is there presently any direct treatment
for the inflammation accompanying acne. The conventional treatment
acts to prevent further problems by alleviating the cause of the
acne; for example, a patient is treated with tetracycline, an
antiobiotic, in hopes of killing the P. acnes, and the death of the
bacteria will effectively end the inflammation and future scarring.
Much as antipyretics, analgesics, decongestants, and antihistamines
have been developed to treat the symptoms of colds and upper
respiratory infections (as opposed to antibiotics and antivirals to
kill off the invading bacteria and viruses), there is a need for
treatments diminishing if not preventing scarring and inflammation
in acne.
SUMMARY OF THE INVENTION
[0007] One object of this invention is to reduce and/or eliminate
scarring in acne-affected skin.
[0008] Another object of this invention is to reduce and/or
eliminate the inflammatory reaction that accompanies acne.
[0009] We have discovered that acne lesions are not only
inflammatory, but that enzymes that degrade the dermal matrix,
including metalloproteinases (MMPs) and other proteases such as
neutrophil elastase, are present in the lesion, and these enzymes
likely cause, or at least significantly contribute, to scarring.
Additionally, the P. acnes products from the lesion are believed to
initiate a signalling cascade which both induces these degradatory
enzymes within the skin and also induces PMNs (polymorphic
leukocytes; e.g., neutrophils) to migrate to the lesion and
contribute to the induction of degradatory enzymes. Still further,
the P. acnes products are believed to cause a signalling cascade
leading to inflammation.
[0010] Our invention is the treatment and prevention of scarring
caused by acne, which treatment and prevention is accomplished
through the administration of a composition comprising an effective
amount of an non-retinoid, non-glucocorticoid inhibitor of a dermal
matrix-degrading enzyme to acne-affected skin. Adminstration may be
topical, systemic (preferably oral), or a combination thereof, and
may be given in combination with another, conventional acne therapy
(e.g., benzoyl peroxide, a retinoid, or a tetracycline). In yet
another embodiment, the topical application of a dermal
matrix-degrading enzyme inhibitor includes the administration of
inhibitors of both MMPs and other proteases. The inhibitor can be a
direct inhibitor, acting specifically on the enzyme, or an indirect
inhibitor, tying up a signalling compound in a pathway leading to
the matrix-degrading enzyme.
[0011] Another aspect of the invention is the administration of a
compound that inhibits the inflammatory reaction and/or the
recruitment of cells resulting in an inflammatory reaction in the
acne lesion. Likewise, this administration can be topical, systemic
(e.g., oral), or a combination thereof. Similarly, this
administration can be accompanied by the co-administration of a
conventional acne therapy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 depicts Western analysis of MMP-8 in human skin in
vivo affected with an acne lesion, in uninvolved skin adjacent to
the lesion.
[0013] FIGS. 2A and 2B are photomicrographs of a skin biopsy of
acne-affected skin and uninvolved skin from the same human
volunteer, where the biopsies are stained to reveal the presence of
neutrophil elastase.
[0014] FIGS. 3A and 3B are photomicrographs of a skin biopsy of
acne-affected skin and uninvolved skin from the same human
volunteer, where the biopsies are stained to reveal the presence of
MMP-1, FIGS. 3A and 3B being taken at a deeper level of the
dermis.
[0015] FIGS. 4A and 4B are color photomicrographs of a skin biopsy
of uninvolved (4A) and acne-affected skin (4B), each having been
stained to reveal the presence of Type I procollagen.
[0016] FIG. 5A is a photomicrograph of a skin biopsy of an acne
lesion stained to reveal the presence of neutrophil elastase, and
FIG. 5B is an in-situ zymogram showing neutrophil collagenase
activity in an acne lesion.
[0017] FIG. 6 is a cartoon depicting a possible mechanism for the
present invention.
[0018] FIGS. 7A-7F are photomicrographs of zymograms of skin
biopsies showing the presence (or absence) of collagenase activity
in uninvolved skin (7A), untreated acne-affected skin (7B),
control-treated acne-affected skin (7C), and in treated
acne-affected skin (7D-7F).
[0019] FIGS. 8A and 8B are graphical representations of data
obtained from biopsies of four individuals with acne-affected skin
examined for the presence of degradative enzymes and inflammatory
signalling compounds.
[0020] FIGS. 9A and 9B are immunohistology photomicrographs of
fluorescently-labelled p65 antibody specific for NF-.kappa..beta.,
FIG. 9A from uninvolved skin showing the staining in the body of
the cells, and FIG. 9B from an acne lesion showing the staining in
the nucleus of the cell.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0021] The matrix of the skin (the dermal matrix), a structural
framework that supports the cells and other structures in the skin,
is comprised of collagen and elastin proteins for structural and
dynamic (elastic) support.
[0022] Scarring of acne-affected skin has been known for a long
time, and the typical treatment philosophy is that curing the acne
will eliminate future scarring. As described in the Background
section, it has also been known that acne includes bacterial
infestation and an inflammatory reaction.
[0023] We have discovered that neutrophils (PMNs), immune cells
that migrate to areas of injury, invade acne-affected skin, and
release both a collagenase (MMP-8) and another protease (neutrophil
elastase) that likely exacerbate scarring. Additionally, we have
discovered that acne-affected skin has an elevated collagenase
(MMP-1) level from resident skin cells that further exacerbates
scarring. By inhibiting these dermal matrix-degrading enzymes,
scarring of acne-affected skin can be lessened. Neutrophils
circulate in the blood and therfore must be recruited by a
signalling mechanism to induce their presence in the skin,
facilitate their infiltration to the affected site, and enable
their release of MMP-8 and elastase. Accordingly, impeding or
disrupting the signalling which induces their presence in the skin
and/or the activity of MMP-8 or elastase is likely to diminish the
accompanying inflammation and the degradatory action of MMP-8
and/or elastase.
[0024] Matrix metalloproteinases (MMPs)-are a family of enzymes
that play a major role in physiological and pathological
destruction of connective tissue, especially collagen. Various
types of collagen and collagenases (types of MMPs) are known in
this field, and a further description can be found in U.S. Pat. No.
5,837,224, and in our co-pending application 89,914, filed 3 Jun.
1998, the disclosures of which are incorporated herein by reference
in their entirety for all purposes. Inhibitors of MMPs (e.g.,
direct inhibitors of the proteinase) and of molecular pathways
(e.g., inhibitors of AP-1 and/or NF-.kappa.B) that affect MMP
expression are known in other fields and likewise are described in
U.S. Pat. No. 5,837,224 and the 89,914 co-pending application.
[0025] MMP-8 preferentially degrades Type I collagen and is a more
active enzyme than MMP-1 and so degrades collagen better.
Neutrophils also release an elastase, a serine protease, an enzyme
that degrades elastin protein in the dermal matrix.
[0026] This invention inhibits scar formation in acne-affected skin
by inhibiting MMPs, especially neutrophil collagenase, generated in
such acne-affected skin, and by inhibiting other dermal
matrix-degrading enzymes. This invention also inhibits the redness
(erythema) and discomfort caused by the inflammatory reaction
accompaning acne.
[0027] For the experiments, the results of which are shown in the
figures described below, human volunteers (each having given
informed consent) were used to assess the differences in
collagenase and elastase concentration between areas of the skin
having an acne lesion and uninvolved adjacent skin areas, and to
evaluate ex vivo the likely effectiveness of certain
inhibitors.
[0028] FIG. 1 depicts a Western blot of MMP-8 collagenase protein
in human skin biopsied from an area having an acne lesion (L in the
figure) and from clear, uninvolved, adjacent areas of skin (C in
the figure) in patients ("PT") 1, 2, and 3. As shown in this
figure, all three patients clearly have MMP-8 present in the acne
lesion, and have no MMP-8 present in unaffected, adjacent areas of
skin.
[0029] FIGS. 2A and 2B are photomicrographs of stained
cross-section biopsies from human dermis and epidermis of a
volunteer's skin. The photographic section on the right (2B) was
taken through an acne lesion, and the one on the left (2A) was
taken from normal, uninvolved skin. Each biopsy section was stained
to show the presence of neutrophil elastase, a matrix-degrading
serine protease. The presence of neutrophil elastase in FIG. 2B
indicates the presence of neutrophils in the skin, presumably due
(directly and/or indirectly) to the acne. The existence of
neutrophil elastase in the skin would be expected to result in a
breakdown of the elastin in the dermal matrix. Thus, we have found
that acne-affected skin contains a significant amount of neutrophil
elastase and that uninvolved skin contains essentially no
neutrophil elastase. This finding indicates that the presence of
acne causes the recruitment and infiltration of immune cells to
acne-affected areas of the skin.
[0030] FIGS. 3A and 3B depict stained cross-sectional biopsies from
a volunteer's acne-affected and uninvolved skin. The biopsies were
taken and stained for the presence of collagenase (MMP-1). FIGS. 3A
and 3B show biopsies from acne-affected (3A) and uninvolved (3B)
skin that were taken from the lower dermis of a volunteer's skin.
In these two figures, stained cells are seen (3A) from
acne-affected skin at the level of the dermis, whereas no staining
is found in the uninvolved skin (3B) at the same level of the
dermis of uninvolved (not acne-affected) skin. Thus, we have found
that acne induces collagenase in the dermis through skin cells
(keratinocytes, fibroblasts) resident in the skin.
[0031] FIGS. 4A and 4B are photomicrographs of stained
cross-sectional biopsies from the skin of a volunteer which have
been stained for the presence of Type I procollagen; FIG. 4A is
from non-acne-affected skin and FIG. 4B is from acne-affected skin.
(The reader is referred to our co-pending application number
285,860, filed 2 Apr. 1999, which describes preventing UV-induced
inhibition of collagen biosynthesis in human skin, and our
co-pending application number 28,435, filed 24 Feb. 1998, regarding
the reduced biosynthesis of procollagen in aged skin, and so are
useful for a further understanding the significance of the lack of
Type I procollagen; the disclosure of those applications is
incorporated herein by reference for all purposes). The presence of
Type I procollagen indicates that dermal cells are producing this
collagen precursor, whereby the dermal matrix is being rebuilt by
new collagen. (Procollagen is made in cells and is soluble; it
passes into the dermal matrix where it is formed into insoluble
collagen.) Uninvolved skin, as shown in FIG. 4A, is producing
procollagen as would be expected, since acne generally starts
affecting people in their teens, and these people typically have
skin that is producing normal, significant amounts of procollagen.
However, as shown in FIG. 4B, skin from the same subject as was
taken for FIG. 4A, there is almost no procollagen production in
acne-involved skin. Thus, we have found that acne-involved skin is
deficient in procollagen production. As we discuss in our prior
applications and patents regarding photoaging, exposure of human
skin to UV radiation induces in that skin MMPs, and it is believed
that imperfect repair of the skin after such MMP-inducing exposure
leads to dermal scarring. Based on the present results, it would
appear that scarring due to acne is significantly exacerbated by
the absence of procollagen in the skin: although the skin is
attempting to heal itself, the lack of procollagen in that skin
means that there is likely to be an imperfect repair. Thus, the
more acne-involved the skin, the less procollagen production, and,
combined with the existence of increased amounts of MMPs in
acne-involved skin, there is a greater probability of imperfect
repair of the acne lesion.
[0032] FIG. 5A depicts acne-involved skin stained for neutrophil
elastase. Neutrophils enter from the blood vessel (off the picture
towards the bottom) and migrate to the surface of the skin (the
epidermis being shown at the top of the panel). The staining is
more significant at the top of the lesion in the dermis, while
there is some staining (black dots) along the bottom portion of the
lesion (suggesting that there are still other neutrophils migrating
to the area). The other panel (FIG. 5B) is an in-situ zymogram
showing the presence of collagenase activity; a section of the acne
lesion is placed on a fluorescently-labelled collagen-coated slide,
and where there is active collagenase that enzyme will destroy the
fluorescently-labelled collagen on the slide and leave a black
background on the panel. As seen in FIG. 5B, there was significant
collagenase activity around the acne lesion. Thus, we have
discovered that in the area of acne lesions there is neutrophil
infiltration with significant elastase and collagenase
activity.
[0033] While not desirous of being constrained to a particular
theory, we believe that scarring due to acne is exacerbated, if not
caused, by defects in skin repair. The skin is continuously trying
to repair itself; in this instance from the degradation caused by
acne. Acne, though more likely the bacterial infestation, leads to
inflammation. The inflammatory response defense mechanism includes
infiltration into the skin of neutrophil immune cells; these cells
generate collagenase and elastase that degrade to dermal matrix,
the degradation products being removed and the matrix then being
repaired. This process of degradation, which is part of the repair
process (i.e., the need to breakdown and remove materials for
further repair and cell growth) is not perfect, and imperfections
or defects in the repaired matrix can be manifest or presented as
scars. Pursuant to the present invention, the administration of an
inhibitor of a dermal matrix-degrading enzyme effective to affect
acne-involved skin inactiviates these destructive proteins or
eliminates their presence by blocking the pathway(s) that creates
or activates them; whether topical or systemic, if the inhibitor is
conveyed to the skin, it will be effective for inhibiting dermal
matrix-degradaing enzymes and thus eliminates their
consequences.
[0034] Again while not desirous of being constrained to a
particular theory, the possible mechanism by which this invention
functions is depicted in the cartoon of FIG. 6. On the left side of
FIG. 6 a hair follicle infected with P. acnes is shown. These
bacteria release LPS (lipopolysaccharide)-like compounds which are
sensed by keratinocytes (KC) (triangles in FIG. 6). (B R Vowels, S
Yang, and J J Leyden, "Induction of proinflammatory cytokines by a
soluble factor of Propionibacterium acnes: implications for chronic
inflammatory acne, Infect. Immun. 1995 63: 3158-3165; the
disclosure of which is incorporated herein by reference). The
toll-like receptor (TLR) family includes LPS receptors, and those
in the keratinocytes are activated by LPS-like products from P.
acnes. Activation of the TLRs causes NF-.kappa.B to enter the cell
nucleus of keratinocyes, as shown in FIGS. 9A and 9B discussed
below. The keratinocytes are thus induced to release chemotactic
factors, especially cytokines (IL-1.beta., IL-8, IL-10,
TNF.alpha.). These factors activate the AP-1 and NF-.kappa.B
pathways, and NF-.kappa.B activates more IL-1 and TNF.alpha. (a
cyclical process; see FIG. 1 in our prior patent U.S. Pat. No.
5,837,224 on photoaging due to UV radiation, the disclosure of
which is incorporated herein by reference). The release of these
factors causes inflammation, including the recruitment of
neutrophils (PMNs; i.e., polymorphonuclear leukocytes) from the
blood supply to the acne lesion; MMP-8 and elastase are preformed
in the neutrophils and so their presence in skin is due to their
presence in neutrophils. As shown in this cartoon, the cytokines
also effect other keratinocytes and fibroblasts (FB), which are
resident in the skin, to generate MMPs. The induction of
matrix-degrading enzymes due to the presence of acne, and the
continual repair of the damage they do, leads to imperfect repair
of the skin. Thus, elimination of the enzymes that degrade the
dermal matrix reduces imperfect repair of the skin, and so lessens
scarring. As shown above in FIGS. 3A-3B, collagenase expression in
acne-affected skin occurs in the dermis. Accordingly, a preferred
composition includes indirect inhibitors of matrix degrading
enzymes, such as glucocorticoids that block recruitment of
neutrophils and other inflammatory immune cells, optionally
retinoids that inhibit MMPs in resident skin cells, and direct
inhibitors of these enzymes, such as serpine (a serine protease
inhibitor analogous to TIMP), all preferably in combination with at
least one compound for treating acne (e.g., benzoyl peroxide or
tetracycline). While retinoids and antibacterials are commonly used
to treat acne, they have not been used in combination with
non-retinoid MMP inhibitors, elastase inhibitors, and/or inhibitors
of the PNM recruitment pathway leading to degradation of the dermal
matrix.
[0035] FIGS. 8A and 8B compare levels of collagen-degrading enzymes
and inflammatory signalling molecules for four individuals, from
their uninvolved and acne-involved skin; for the acne-involved
skin, each of the volunteers is represented by a differently-shaped
data point (square, circle, diamond, triangle). FIG. 8A shows the
change in the amount of mRNA encoding for three different dermal
matrix-degrading enzymes (MMP-1, MMP-3, and MMP-9) between
uninvolved (UNINV) and acne-affected (ACNE) for these individuals;
the dashed horizontal line represents the mean value; and the three
different scales should be noted. In general, FIG. 8A shows that in
acne-affected skin, MMP-1 is elevated an average of over 500 times
from uninvolved skin, MMP-3 is elevated an average of over 1000
times from uninvolved skin, and MMP-9 is elevated an average of
almost 15 times compared with uninvolved skin. FIG. 8B shows the
difference in the amount of mRNA encoding for the inflammatory
cytokines mentioned above (TNF.alpha., IL-1.beta., IL-8, and IL-10)
between uninvolved and acne-affected skin; as with FIG. 8A, each of
the individuals is represented by a differently-shaped data point,
and the mean value is shown as the horizontal dashed line. As with
the collagen-degrading enzymes, the amounts of mRNA encoding the
cytokines increased from ininvolved to acne-affected skin:
TNF.alpha. was about four times higher, IL-1.beta. was over 25
times higher, IL-8 was over 5000 times higher, and IL-10 was about
75 times higher. These data confirm that increased cytokine
concentrations (inferred from an increase in their mRNA levels) are
present in acne lesions, and thus the scarring and collagen
degradation due to acne can be treated with a combination of MMP
inhibitors and cytokine inhibitors.
[0036] FIGS. 9A and 9B compare the location of fluorescent
p65-labelled NF-.kappa..beta. in uninvolved and acne-involved human
skin biopsies via immunohistological techniques; p65 antibody forms
a complex with NF-.kappa..beta., and the fluorescent labelling
allows the location of the antibody to be visualized. The biopsy in
FIG. 9A shows that any NF-.kappa..beta. present in the epidermis is
present in the body of the cells; the insert is a close-up showing
that the nuclei of the cells (dark objects) are surrounded by the
cell body in which the fluorescently-p65-labelled NF-.kappa..beta.
resides (red area). The treatment offered in this application
involves, as shown in FIG. 6, activation of cells by
NF-.kappa..beta.. FIG. 9B, taken through an acne lesion, shows that
the NF-.kappa..beta. has entered the cell nucleus of keratinocytes
throughout the follicular epidermis involved in the lesion, with
the staining being somewhat reduced in the remaining epidermis; the
insert shows that the NF-.kappa..beta. has entered the cells'
nuclei (hence the red fluorescence is ovoid) and that the cell body
is no longer stained by the presence of NF-.kappa..beta.. These
figures thus show that there is prominent nuclear localization of
NF-.kappa..beta. in acne-involved human skin lesions in vivo as
compared with uninvolved human skin. This nuclear localization is
consistent with the mechanism proposed in FIG. 6 (but not relied
upon for patentability), that NF-.kappa..beta. is present in the
inflammatory signalling associated with acne lesions.
[0037] In light of our findings, acne-affected patients can be
helped by decreasing the activity of matrix-degrading enzymes in
the area of acne lesions. This can be accomplished by various means
which are not mutually exclusive. One method is to disrupt the
signalling caused by P. acnes byproducts that results in cytokines
and MMPs. Another method is to disrupt the signalling that results
in the recruitment of neutrophils with the accompanying neutrophil
elastase and collagenase. Our present results suggest the mechanism
shown in FIG. 6, a cartoon depicting the signalling in acne
lesions. The P. acnes products induce keratinocytes (KC) to produce
tumor necrosis factor alpha (TNF.alpha.), interlukin-1.beta.
(IL-1.beta.), and interlukins-8 and 10 (IL-8, IL-10), all
cytokines. These cytokines induce resident skin cells to produce
MMPs which degrade the dermal matrix. They also cause inflammation
(e.g., redness, vasodilation, etc.) which is a signal for
recruitment of neutrophils containing collagenase and elastase to
the acne lesion; the neutrophil collagenase and the elastase
contribute to degradation of the dermal matrix. The lack of
procollagen biosynthesis, as shown in FIG. 4B, contributes to
imperfect repair of the matrix. The end result is scarring.
[0038] To thwart the apparently inevitable result of acne scarring
and inflammation, this invention disrupts the signalling pathways.
More particularly, this invention uses an non-retinoid, non-steriod
topically-applied composition, optionally in combination with a
retinoid and/or a steriod, to inhibit this signalling, whereby
degradation of the matrix is decreased and procollagen biosynthesis
is restored, allowing the skin to heal with less scarring and less
inflammation. Aspirin and E5510 (described by Fujimori, T., et at.,
Jpn J Pharmacol (1991) 55(1):81-91) inhibit NF-.kappa.B activation.
Farnesyl transferase inhibitors such as B-581 (described by Garcia
A. M., et al., J Biol Chem (1993) 268(25):18415-18), BZA-5B
(described by Dalton M. B. et al., Cancer Res (1995)
55(15):3295-3304), farnesyl acetate, and (.alpha.-hydroxyfarnesyl)
phosphoric acid act on RAS and thus inhibit activation of the
resultant ERK cascade; ERK leads to c-fos, which heterodimerizes
with c-jun to create AP-1. Other useful inhibitors are those that
inhibit NF-.kappa.B, such as sulfasalazine and parthenolide, serine
protease (elastase) inhibitors, and antiadhesion molecules such as
neutrophil infiltration inhibitors (e.g., selectin antagonists). As
described in the aforementioned applications relating to
UV-induction of MMPs, we have shown that so-called "antioxidants",
like N-acetylcysteine (NAC), are useful at inhibiting MMPs, and
have been shown in the literature (discussed below) to inhibit
AP-1, NF-.kappa.B, and IL-8. Because the signalling that we have
identified (which contributes to scarring and inflammation in acne)
appears similar to the signalling by which UV irradiation induces
MMPs, similar "antioxidants" as disclosed in those application and
discussed below (e.g., NAC, FDO) are likely to be useful for
combatting acne scarring and inflammation.
[0039] As used herein, "inhibitors" of MMPs and other dermal
matrix-degrading enzymes, such as elastase, inhibit one or more of
the steps in the natural physiological pathways leading to the
production of these enzymes and/or directly inhibit one or more of
these proteases, or they directly inhibit the activity of the
enzyme. Thus, as used herein an "inhibitor" excludes retinoids,
inasmuch as retinoids and tetracyclines have been known for
treating acne, this invention is directed to the novel use of a
non-retinoid enzyme inhibitor, which use may be combined with the
conventional use of a retinoid and/or a tetracycline. Thus, an
"inhibitor" is a non-retinoid compound that directly inhibits one
or more dermal matrix-degrading enzymes and/or indirectly inhibits
the enzyme by inhibiting some portion of an upstream pathway(s)
leading to one or more of these dermal matrix-degrading enzymes.
Inhibition of the upstream pathway of these dermal matrix-degrading
enzymes includes inhibition of one or more of the various
signalling compounds and/or of the transcription factors (e.g.,
NF-.kappa.B, or cJUN and cFOS which together create AP-1) by which
these enzymes are produced naturally.
[0040] MMPs are also inhibited by BB2284 (described by Gearing, A.
J. H. et al., Nature (1994) 370:555-557), GI129471 (described by
McGeehan G. M., et al., Nature (1994) 370:558-561), and TIMPs
(tissue inhibitors of metalloproteinases, which inhibit vertebrate
collagenases and other metalloproteases, including gelatinase and
stromelysin). Other compounds useful for the present invention are
direct MMP inhibitors such as hydroxamate and hydroxy-urea
derivatives, the latter exemplified by Galardin, Batimastat, and
Marimastat, and those disclosed in EP-A1-0 558635 and EP-A1-0
558648 (disclosed as useful for inhibiting MMPs in the treatment
of, among other etiologies, skin ulcers, skin cancer, and
epidermolysis bullosa).
[0041] Indirect MMP inhibitors include kinase inhibitors genistein
and quercetin (as described in U.S. Pat. Nos. 5,637,703, 5,665,367,
and FR-A-2,671,724, the disclosures of which are incorporated
herein by reference) and related compounds, as well as other
antioxidants such as NAC (N-acetyl cysteine), discussed below.
Still further, other kinase inhibitors are SB202190 (described by
Lee, J. C., et al., Nature (1994) 372:739-746) and PD98059
(described by Dudley, D. T., et al., PNAS (USA) (1995)
92:7686-7689) inhibit specific kinases in the cascades, geranyl
geranyltransferase inhibitors and lisofylline, which inhibit
activation of the JNK cascade resulting from RAC/CDC42 activation,
and U0126
(1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene). As
noted above, compounds that inhibit cytokines are indirect MMP
inhibitors because interrupting the signalling pathway effectively
inhibits MMPs. Compounds such as
[0042] As mentioned, various compounds termed "antioxidants" are
also useful as MMP inhibitors. While not desirous of being
constrained to any particular theory of operation, these compounds
may quench or otherwise reduce free radicals and reactive oxygen
species which may initiate or lead to MMP induction, such as via
the MAP kinase cascade. These compounds include glutathione and its
precursors, such as N-acetyl cysteine (NAC) or glutathione ethyl
ester, more broadly N--CH.sub.3(CH.sub.2).sub.nCO cysteine (wherein
n is an integer from zero to eight, more preferably not more than
4), and related compounds and derivatives thereof as described in
U.S. Pat. No. 5,296,500 (the disclosure of which is incorporated
herein by reference). These other MMP inhibitors include
water-soluble compounds such as vitamin C and NAC, and FDO. Various
other compounds that may act as MMP inhibitors include:
lipid-soluble compounds such as .beta.-carotene and its derivatives
or other carotenoids; glutathione and derivatives thereof (or of
NAC); .alpha.-lipoic acid (1,2-dithiolane-3-pentanoic acid);
selenium compounds such as Ebselen
(2-phenyl-1,2-benzisoselenazol-3(2H)-one); isoflavones such as
genistein (isoflavone), quercetin (flavon-3-ol), and pycnogenol
(flavan-3-ol(s)); ergothioneine; saponin (e.g., from Polypodium
leucotomos); ginkgo biloba extract (flavoneglycoside and
terpenelactone) and fever few (Chrysanthemum parthenium) extract
(sesquiterpene lactone).
[0043] Inhibitors of activator protein-1 (AP-1) are likely to
inhibit the subsequent signalling that results in the presence of
MMPs in the dermal matrix; the more of the pathway that is
inhibited, the more likely there will be no induction of MMPs.
Among various compounds that have been found to inhibit AP-1 and
may likely be used topically include the following. Cannabinoids:
Faubert and Kaminski; "AP-1 activity is negatively regulated by
cannabinol through inhibition of its protein components, c-fos and
c-jun", J Leukoc Biol, vol. 67, no. 2 (2000 February, pp. 259-66)
(Cannabinoid compounds exhibit immunosuppressive actions that are
putatively mediated through Gi-protein coupled receptors that
negatively regulate adenylate cyclase. However, recent studies
suggest that cannabinoids modulate other signaling cascades.
Cannabinol inhibited binding to AP-1-containing sites from the
interleukin-2 promoter, in part, due to decreased nuclear
expression of c-fos and c-jun. Thus, cannabinoid-induced
immunosuppression involves disruption of the ERK signaling
cascade.) Deferroxamine (DFO); Kramer-Stickland et al., "Inhibitory
effects of deferoxamine on UVB-induced AP-1 transactivation",
Carcinogenesis, vol. 20, no. 11, November 1999, pp. 2137-42
(Production of reactive oxygen species (ROS) by iron can contribute
directly to DNA and protein damage and may contribute to cell
signaling and proliferation. DFO treatment 24 h prior to UVB
irradiation reduced UVB-induced AP-1 transactivation by
approximately 80%, with the effect of DFO diminishing as
pre-treatment time was shortened. Treatment with FeCl(3) a minimum
of 6 h prior to UVB potentiated the UVB induction of AP-1
transactivation by 2-3-fold.) Separately, gadolinium chloride and
alpha-tocopherol: Camandola et al., "Liver AP-1 activation due to
carbon tetrachloride is potentiated by 1,2-dibromoethane but is
inhibited by alpha-tocopherol or gadolinium chloride", Free Radic
Biol Med, vol. 26, no. 9-10, May 1999, pp. 1108-16. Cyclosporin A:
Sugano et al., "Cyclosporin A inhibits collagenase gene expression
via AP-1 and JNK suppression in human gingival fibroblasts, J
Periodontal Res, vol. 33, no. 8, November 1998, pp. 448-452
(Cyclosporin A is able to affect signal transduction of
lipidpolysaccharide-induced collagenase expression in fibroblasts;
treatment of fibroblasts with LPS caused activation of collagenase
gene, activator protein-1 (AP-1) and c-Jun N-terminal kinase (JNK).
These activations were blocked by CsA. They suggest that inhibitory
effects of CsA on LPS-induced signal transduction may contribute to
the mechanism of CsA-induced gingival overgrowth. Catachins:
Barthelman et al., "(-)-Epigallocatechin-3-gallate inhibition of
ultraviolet B-induced AP-1 activity", Carcinogenesis, vol. 19,
no.12, Dec. 1998, pp. 22014 (using cultured human keratinocytes,
UVB-induced AP-1 activity is inhibited by EGCG in a dose range of
5.45 nM to 54.5 microM; EGCG is effective at inhibiting AP-1
activity when applied before, after or both before and after UVB
irradiation; EGCG also inhibits AP-1 activity in the epidermis of a
transgenic mouse model). Naphthopyranomycins and exfoliamycins,
such as K1115 A (Naruse et al., "K1115A, a new anthraquinone that
inhibits the binding of activator protein-1 (AP-1) to its
recognition sites. II. Taxonomy, fermentation, isolation,
physico-chemical properties and structure determination," J
Antibiot (Tokyo), vol 51, no. 6, June 1998, pp. 545-52; the
anthraquinone 3,8-dihydroxy-1-propylanthraquinone-2-carboxylic
acid). DHEA: Dashtaki et al., "Dehydroepiandrosterone and analogs
inhibit DNA binding of AP-1 and airway smooth muscle
proliferation", J Pharmacol Exp Ther, vol. 285, no. 2, 1998 May
(pp. 876-83) (dehydroepiandrosterone (DHEA) and its analogs such as
16-alpha-bromoepiandrosterone). Oleanolic acid glycosides: Lee et
al., "Momordins inhibit both AP-1 function and cell proliferation,"
Anticancer Res, vol. 18, no. 1A, January-February 1999 (pp.
119-24). Monoterpene perillyl alcohol: Barthelman et al.,
"Inhibitory effects of perillyl alcohol on UVB-induced murine skin
cancer and AP-1 transactivation", Cancer Res., vol. 58, no. 4, 15
February 1998 (pp. 711-6). Curcumin, which inhibits both AP-1 and
NF-.kappa.B: Xu et al., "Curcumin inhibits ILl alpha and TNF-alpha
induction of AP-1 and NF-kB DNA-binding activity in bone marrow
stromal cells," Hematopathol Mol Hematol, vol. 11, no. 1, 1997-8
(pp. 49-62); and Pendurthi et al., "Suppression of activation of
transcription factors Egr-1, AP-1, and NF-kappa B," Arterioscler
Thromb Vasc Biol, vol. 17, no.12, December 1997 (pp. 3406-13); and
Bierhaus et al., "The dietary pigment curcumin reduces endothelial
tissue factor gene expression by inhibiting binding of AP-1 to the
DNA and activation of NF-kappa B," Thromb Haemost, vol. 77, no.
4,1997 April (pp. 772-82). Aspirin (acetylsalicylic acid): Huang et
al., "Inhibition of ultraviolet B-induced activator protein-1
(AP-1) activity by aspirin in AP-1-luciferase transgenic mice", J
Biol Chem, vol. 272, no. 42,17 October 1997 (pp. 26325-31).
Pyrrolidine dithiocarbamate and N-acetyl cysteine (inhibit AP-1,
NF-.kappa.B, and IL-8): Munoz et al., "Pyrrolidine dithiocarbamate
inhibits the production of interleukin-6, interleukin-8, and
granulocyte-macrophage colony-stimulating factor by human
endothelial cells in response to inflammatory mediators: modulation
of NF-kappa B and AP-1 transcription factors activity", Blood, vol.
88, no. 9, 1996 Nov 1 (pp. 3482-90). Metal salts, such as gold (I)
and selenite: Handel et al., "Inhibition of AP-1 binding and
transcription by gold and selenium involving conserved cysteine
residues in Jun and Fos," Proc Natl Acad Sci USA, vol. 92, no. 10,
1995 May 9 (pp. 4497-501) (in electrophoretic mobility-shift
analyses, AP-1 DNA binding was inhibited by gold(I) thiolates and
selenite, with 50% inhibition occurring at approximately 5 microM
and 1 microM, respectively; and other metal ions inhibited at
higher concentrations, in a rank order correlating with their thiol
binding affinities); and Spyrou et al., "AP-1 DNA-binding activity
is inhibited by selenite and selenodiglutathione", FEBS Lett, vol.
368, no. 1, 1995 Jul 10 (pp. 59-63) (selenite and
selenodiglutathione (GS-Se-SG)); and Williams et al.,
"Aurothioglucose inhibits induced NF-kB and AP-1 activity by acting
as an IL-1 functional antagonist", Biochim Biophys Acta, vol. 1180,
no. 1,1992 Oct 13 (pp. 9-14).
[0044] Elastase inhibitors include procyanidines and
proanthocyanidines, which non-competitively inhibit the activities
of the proteolytic enzymes collagenase (IC.sub.50=38 nmol/l) and
elastase (IC.sub.50=4.24 nmol/l) (Arzneimittelforschung (GERMANY)
May 1994, 44 (5) p592-601), N-acetyl cysteine (e.g., U.S. Pat. No.
5,637,616 for a disclosure of NAC as an inhibitor of proteases that
result in mucosal or skin lesions) and derivatives thereof (as
described in our copending application 89,914, filed 3 Jun. 1998
(the disclosure of which is incorporated herein by reference)).
Additional elastase inhibitors are described in the following
disclosures.
6-Acylamino-2-(alkylsulfonyl)oxy-1H-isoindole-1,3-dione and related
diones: Kerrigan et al.,
"6-Acylamino-2-(alkylsulfonyl)oxy-1H-isoindole-1,3-dione
mechanism-based inhibitors of human leukocyte elastase", Bioorg Med
Chem Lett, vol. 10, no. 1, 2000 Jan 3 (pp. 27-30) (acylamino
substitution in the 6-position increases selectivity and potency of
these inhibitors for human leukocyte elastase); Gutschow et al.,
"2-(diethylamino)thieno-1,3-oxazin4-ones as stable inhibitors of
human leukocyte elastase", J Med Chem, vol. 42, no. 26, 1999 Dec 30
(pp. 5437-47) (2-(diethylamino)thieno[1,3]oxazin-4-one). Caffeic
acid derivatives: Melzig et al., Inhibition of granulocyte elastase
activity by caffeic acid derivatives", Pharmazie, vol. 54, no. 9,
1999 Sep (pp. 712). Pyridyl esters of benzopyrans: Doucet et al.,
"6-Substituted 2-oxo-2H-1-benzopyran-3-carboxylic acid as a core
structure for specific inhibitors of human leukocyte elastase", J
Med Chem, vol. 42, no. 20, 1999 Oct 7 (pp. 4161-71). Certain
beta-lactams: Taylor et al., "Novel mechanism of inhibition of
elastase by beta-lactams is defined by two inhibitor crystal
complexes", J Biol Chem, vol. 274, no. 35, 1999 Aug 27 (pp.
24901-5) (the presence of a hydroxyethyl substituent on the
beta-lactam ring provides a new reaction pathway resulting in the
elimination of the hydroxyethyl group and the formation of a
stabilizing conjugated double bond system); Wilmouth et al.,
"Inhibition of elastase by N-sulfonylaryl beta-lactams: anatomy of
a stable acyl-enzyme complex", Biochemistry, vol. 37, no. 50, 1998
Dec 15 (pp. 17506-13); pyrrolidone trans-lactams and
trans-lactones) such as disclosed by Macdonald et al., "Syntheses
of trans-5-oxo-hexahydro-pyrrolo 3,2-bpyrroles and
trans-5-oxo-hexahydro-furo 3,2-b-pyrroles (pyrrolidine
trans-lactams and trans-lactones): new pharmacophores for elastase
inhibition", J Med Chem, vol. 41, no. 21,1998 Oct 8 (pp. 3919-22).
Benzoyl aminoacetic acid derivatives: Sakuma et al., "ONO-5046 is a
potent inhibitor of neutrophil elastase in human pleural effusion
after lobectomy", Eur J Pharmacol, vol. 10 353, no. 2-3,1998 Jul 24
(pp. 273-9) (sodium
N-2-4-(2,2-Dimethylpropionyloxy)phenyl-sulfonylamino-benzoyl-aminoacetic
acid). Complex sulfates: Fujie et al., "Release of neutrophil
elastase and its role in tissue injury in acute inflammation:
effect of the elastase inhibitor, FR134043", Eur J Pharmacol, vol.
374, no. 1,1999 Jun 11 (pp. 117-25)
(disodium-(Z,1S,15S,18S,24S,27R,29S,34S,37R)-29-benzyl-21-ethylidene-27-h-
ydroxy-15-isobutyrylamino-34-isopropyl-31,
37-dimethyl-10,16,19,22,30,32,35,38-octaoxo-36-oxa-9,11,17,20,23,28,31,33-
-octaazatetracyclo
16,13,6,1(24,28)0(3,8)-octatriconta-3,5,7-trien-5,6-diyl disulfate.
Azaisochromens: Mitsuhashi et al., "Pharmacological activities of
TEI-8362, a novel inhibitor of human neutrophil elastase", Br J
Pharmacol, vol. 126, no. 5, 1999 Mar (pp. 1147-52)
(4-(N-(3-((3-carboxypropyl)amino)-8-methyl-1-oxo-4-azaisochromen-6-yl)car-
bamoyl)-4-((phenyl-methoxy)carbonylamino)butanoic acid
(C.sub.26H.sub.28N.sub.4O.sub.9)). Acetamides: Yamano et al.,
"Protective effects of a PAF receptor antagonist and a neutrophil
elastase inhibitor on multiple organ failure induced by cerulein
plus lipopolysaccharide in rats", Naunyn Schmiedebergs Arch
Pharmacol, vol. 358, no. 2,1998 Aug (pp. 253-63)
(2-(3-methylsulfonylamino-2-oxo-6-phenyl-1,2-dihydro-1-pyridyl)-N-
-(3,3,3-trifluoro-1-isopropyl-2-oxopropyl)acetamide). Molecules
having only a few amino acid residues which are effective for
penetrating the skin: Yamano et al., "Protective effect of a
pancreatic elastase inhibitor against a variety of acute
pancreatitis in rats" Jpn J Pharmacol, vol. 77, no. 3, 1998 Jul
(pp. 193-203) (trifluoroacetyl-L-lysyl-L-alaninanilide
hydrochloride). Trifluoromethyl ketones: Huang et al., "Effect of
trifluoromethyl ketone-based elastase inhibitors on neutrophil
function in vitro", J Leukoc Biol, vol. 64, no. 3,1998 Sep (pp.
322-30) (new family of elastase inhibitors IC1200355 and ZD0892).
Sulfone derivatives of thiazolidine-3-ones: Groutas et al., "Potent
and specific inhibition of human leukocyte elastase, cathepsin G
and proteinase 3 by sulfone derivatives employing the
1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold", Bioorg Med Chem,
vol. 6, no. 6, 1998 Jun (pp. 661-71). Peptidyl
trifluoromethylalcohols: Amour et al., "Stereoselective synthesis
of peptidyl trifluoromethyl alcohols and ketones: inhibitory
potency against human leucocyte elastase, cathepsin G, porcine
pancreatic elastase and HIV-1 protease", J Pharm Pharmacol, vol.
50, no. 6,1998 Jun (pp. 593-600) (beta-peptidyl trifluoromethyl
alcohols (TFMAs) Z-L-Val-NH-*CH(Y)*CH(OH)--CF.sub.3, where *C is
the chiral centre, varied in the nature of the substituent Y, a
phenylethyl --(CH.sub.2).sub.2--C.sub.6H.sub.5 or an isopropyl
--CH(CH.sub.3).sub.2 group; phenylethyl had IC.sub.50=15 .mu.M,
whereas isopropyl had IC.sub.50=200 .mu.M). Benzoylamino acetates:
Shinguh et al., "Biochemical and pharmacological characterization
of FK706, a novel elastase inhibitor", Eur J Pharmacol, vol. 337,
no. 1,1997 Oct 15 (pp. 63-71) (FK706, sodium
2-4-(S)-1-(S)-2-(RS)-3,3,3-trifluoro-1-isopropyl-2-oxopropyl-aminocarbony-
l-pyrrolidin-1-yl-carbonyl-2-methylpropyl-aminocarbonyl-benzoylamino
acetate, C.sub.26H.sub.32F.sub.3N.sub.4NaO.sub.7, a synthetic
water-soluble inhibitor of human neutrophil elastase).
Cephalosporin derivatives: Rees et al., "Inhibition of neutrophil
elastase in CF sputum by L-658,758", J Pharmacol Exp Ther, vol.
283, no. 3, 1997 Dec (pp. 1201-6); Buynak et al.,
"7-alkylidenecephalosporin esters as inhibitors of human leukocyte
elastase", J Med Chem, vol. 40, no. 21,1997 Oct 10 (pp. 3423-33)
(7-alkylidene, 7-haloalkylidene, and 7-cyanomethylidene benzhydryl
ester 7-(cyanomethylidene)cephalosporin sulfone derivatives).
Azabicyclic compounds and perhydroindoles: Portevin et al., "Dual
inhibition of human leukocyte elastase and lipid peroxidation: in
vitro and in vivo activities of azabicyclo 2.2.2-octane and
perhydroindole derivatives", J Med Chem, vol. 40, no. 12,1997 Jun
6, (pp. 1906-18) (selective human leukocyte elastase (HLE)
inhibitors of the Val-Pro-Val type in which the central proline
residue was replaced by normatural amino acids Phi
((2S,3aS,7aS)-perhydroindole-2-carboxylic acid) and
Abo((3S)-2-azabicyclo-2.2.2-octane-3-carboxylic acid).
Trialkylammonium salts: Kouadri-Boudjelthia and Wallach,
"Hydrophobic interactions are involved in the inhibition of human
leukocyte elastase by alkyltrimethylammonium salts", Int J Biochem
Cell Biol, vol. 29, no. 2, 1997 Feb (pp. 353-9) (preferably alkyl
chain longer than ten carbon atoms). Pivaloyloxy benzene
derivatives: Imaki et al., "Non-peptidic inhibitors of human
neutrophil elastase: the design and synthesis of
sulfonanilide-containing inhibitors", Bioorg Med Chem, vol. 4, no.
12, 1996 Dec. (pp. 2115-34) (sulfonanilide-containing analogues
most promising). Functionalized N-aryl azetidin-2-ones: Joyeau et
al., "Synthesis and inhibition of human leucocyte elastase by
functionalized N-aryl azetidin-2-ones: effect of different
substituents on the aromatic ring", J Pharm Pharmacol, vol. 48,
no.12,1996 Dec. (pp. 1218-30) (N-aryl-3,3-difluoroazetidin-2-ones
featured by a latent electrophilic methylene quinoniminium moiety,
and incorporate on their aromatic ring either an alkyl moiety, a
methoxy substituent or a carboxylic group; some proved to be good
inactivators of human leucocyte elastase). Saccharine derivatives:
Groutas et al., "Design, synthesis, and in vitro inhibitory
activity toward human leukocyte elastase, cathepsin G, and
proteinase 3 of saccharin-derived sulfones and congeners", Bioorg
Med Chem, vol. 4, no. 9,1996 Sep. (pp. 1393-400) (derivatives has
sulfinate leaving group; inhibitory activity is dependent on the
nature and pKa of the leaving group, and the synthesized saccharin
derivatives exhibit selective inhibition toward HLE).
Mucopolysaccharides, such as heparin: Volpi, "Inhibition of human
leukocyte elastase activity by heparins: influence of charge
density", Biochim Biophys Acta, vol. 1290, no. 3, 1996 Aug 13 (pp.
299-307) (heparins strongly inhibit elastase activity, and there is
a significant linear dependence between charge density
(sulfate-to-carboxyl ratio) and enzymatic activity).
Exopolysaccharides: Ying et al., "Alginate, the slime
exopolysaccharide of Pseudomonas aeruginosa, binds human leukocyte
elastase, retards inhibition by alpha 1-proteinase inhibitor, and
accelerates inhibition by secretory leukoprotease inhibitor", Am J
Respir Cell Mol Biol, vol. 15, no. 2, 1996 Aug. (pp. 283-91) (data
support a model in which each elastase molecule interacts with a
total of 19 uronic acid units on the alginate, primarily through
electrostatic forces).
[0045] NF-.kappa.B inhibitors include those disclosed in the
following references. Cyclopentenone prostaglandins: Rossi et al.,
"Anti-inflammatory cyclopentenone prostaglandins are direct
inhibitors of IkappaB kinase", Nature, vol. 403, no. 6765, 2000 Jan
6 (pp. 103-8). Quercetin and staurosporine: Peet and Li, "IkappaB
kinases alpha and beta show a random sequential kinetic mechanism
and are inhibited by staurosporine and quercetin", J Biol Chem,
vol. 274, no. 46, 1999 Nov 12 (pp. 32655-61) (but not the quercetin
analogue Daidzein). Nepalolide A: Wang et al., "Nepalolide A
inhibits the expression of inducible nitric oxide synthase by
modulating the degradation of IkappaB-alpha and IkappaB-beta in C6
glioma cells and rat primary astrocytes", Br J Pharmacol, vol. 128,
no. 2,1999 Sep. (pp. 345-56). Turmeric (curcumin): Plummer et al.,
"Inhibition of cyclo-oxygenase 2 expression in colon cells by the
chemopreventive agent curcumin involves inhibition of NF-kappaB
activation via the NIK/IKK signalling complex", Oncogene, vol. 18,
no. 44, 1999 Oct 28 (pp. 6013-20). Salicylates: Stevenson et al.,
"Salicylic acid and aspirin inhibit the activity of RSK2 kinase and
repress RSK2-dependent transcription of cyclic AMP response element
binding protein- and NF-kappa B-responsive genes", J Immunol, vol.
163, no. 10, 1999 Nov 15 (pp. 5608-16). Diterpenes: de las Heras et
al., "Inhibition of NOS-2 expression in macrophages through the
inactivation of NF-kappaB by andalusol", Br J Pharmacol, vol. 128,
no. 3,1999 Oct (pp. 605-12) (andalusol,
ent-6a,8a,18-trihydroxy-13(16),14-labdadiene, is a naturally
occurring diterpene, isolated from Sideritis foetens (Lamiaceae).
N-substituted benzamides: Liberg et al., "N-substituted benzamides
inhibit NFkappaB activation and induce apoptosis by separate
mechanisms", Br J Cancer, vol. 81, no. 6,1999 Nov (pp. 981-8).
While not preferred due to potential toxicity issues, arsenic:
Estrov et al., "Phenylarsine oxide blocks
interleukin-1.beta.-induced activation of the nuclear transcription
factor NF-.kappa.B, inhibits proliferation, and induces apoptosis
of acute myelogenous leukemia cells", Blood, vol. 94, no. 8, 1999
Oct 15 (pp. 2844-53). Genistein: Tabary et al., "Genistein inhibits
constitutive and inducible NFkappaB activation and decreases IL-8
production by human cystic fibrosis bronchial gland cells", Am J
Pathol, vol. 155, no. 2,1999 Aug (pp. 473-81). Theophylline: Tomita
et al., "Functional assay of NF-kappaB translocation into nuclei by
laser scanning cytometry: inhibitory effect by dexamethasone or
theophylline", Naunyn Schmiedebergs Arch Pharmacol, vol. 359, no.
4, 1999 Apr (pp. 249-55). Cepharanthine: a plant alkaloid (I)
(Merck Index 11, 306,1981), and described in U.S. Pat. Nos.
2,206,407 and 2,248,241, and Japanese Patents 120,483, 128,533, and
141,292. Trifluoroalkyl salicylates: Bayon et al.,
"4-trifluoromethyl derivatives of salicylate, triflusal and its
main metabolite 2-hydroxy-4-trifluoromethylbenzoic acid, are potent
inhibitors of nuclear factor kappaB activation", Br J Pharmacol,
vol. 126, no. 6, 1999 Mar (pp. 1359-66)
(2-hydroxy-4-trifluoromethylbenzoic acid (HTB) and
2-acetoxy4-trifluoromethylbenzoic acid (triflusal), both more
potent than aspirin or salicylate as inhibitors of NF-.kappa.B,
indicating that the incorporation of a 4-trifluoromethyl group to
the salicylate molecule strongly enhances its inhibitory effect on
NF-.kappa.B activation). Quinapril: quinapril hydrochloride is
chemically described as
[3S-[2[R*(R*)],3R*]]-2-[2-[[1-(ethoxycarbonyl)-3-phenylpropyl]amino]-1-ox-
opropyl]-1,2,3,4-tetrahydro-3-isoquinolinecarboxylic acid,
monohydrochloride. Its empirical formula is
C.sub.25H.sub.30N.sub.2O.sub.5.HCl. Cyclosporine A: Meyer et al.,
"Cyclosporine A is an uncompetitive inhibitor of proteasome
activity and prevents NF-kappaB activation", FEBS Lett, vol. 413,
no. 2, 1997 Aug 18 (pp. 354-8). Arachidonic acid derivatives:
Thommensen et al., "Selective inhibitors of cytosolic or secretory
phospholipase A2 block TNF-induced activation of transcription
factor nuclear factor-kappa B and expression of ICAM-1", J Immunol,
vol. 161, no. 7, 1998 Oct 1 (pp. 3421-30) (TNF-induced activation
of NF-.kappa.B inhibited by trifluoromethyl ketone analogue of
arachidonic acid (AACOCF.sub.3), methyl arachidonyl
fluorophosphate, trifluoromethyl ketone analogue of
eicosapentaenoic acid (EPACOCF.sub.3), 12-epi-scalaradial, and
LY311727; arachidonyl methyl ketone analogue (AACOCH.sub.3) and the
eicosapentanoyl analogue (EPACHOHCF.sub.3) had no effect on
TNF-induced NF-.kappa.B activation. Genistein, erbstatin: Natarajan
et al., "Protein tyrosine kinase inhibitors block tumor necrosis
factor-induced activation of nuclear factor-KB, degradation of
I.kappa.B.alpha., nuclear translocation of p65, and subsequent gene
expression", Arch Biochem Biophys, vol. 352, no. 1, 1998 Apr 1 (pp.
59-70). Fasudil: 1-(5-isoquinolinesulfonyl)homopiperazine
hydrochloride (fasudil hydrochloride); Sato et al., "Inhibition of
human immunodeficiency virus type 1 replication by a bioavailable
serine/threonine kinase inhibitor, fasudil hydrochloride", AIDS Res
Hum Retroviruses, vol. 14, no.4, 1998 Mar 1 (pp. 293-8). ACE
(angiotensin converting enzyme) inhibitors, like quinipril:
Hernandez-Presa et al., "Angiotensin-converting enzyme inhibition
prevents arterial nuclear factor-kappa B activation, monocyte
chemoattractant protein-1 expression, and macrophage infiltration
in a rabbit model of early accelerated atherosclerosis",
Circulation, vol. 95, no. 6, 1997 Mar 18 (pp. 153241). Synthetic
1,3,7-trialkyl xanthine derivatives, such as pentoxifylline
(3,7-dimethyl-1-(5-oxohexyl)xanthine; Drugs & Aging 1995, 7/6:
480-503) and denbufylline (1,3-dibutyl-7-(2-oxopropyl)xanthine);
Lee et al., "Pentoxifylline blocks hepatic stellate cell activation
independently of phosphodiesterase inhibitory activity", Am J
Physiol, vol. 273, no. 5 Pt 1, 1997 Nov (pp. G1094-100).
Benzophenanthradine derivatives: Chaturvedi et al., "Sanguinarine
(pseudochelerythrine) is a potent inhibitor of NF-.kappa.B
activation, I.kappa.B.alpha. phosphorylation, and degradation", J
Biol Chem, vol. 272, no. 48, 1997 Nov 28 (pp. 30129-34)
(sanguinarine, a benzophenanthridine alkaloid). Actinomycin D:
Faggioli et al., "Protein synthesis inhibitors cycloheximide and
anisomycin induce interleukin-6 gene expression and activate
transcription factor NF-.kappa.B", Biochem Biophys Res Commun, vol.
233, no. 2, 1997 Apr 17 (pp. 507-13) (IL-6 mRNA accumulation in two
human cell lines, MDA-MB-231 and HeLa, stimulated by cycloheximide
or anisomycin is almost completely inhibited in the presence of
actinomycin D). Hydroxyanthranilic acids: Sekkai et al.,
"Inhibition of nitric oxide synthase expression and activity in
macrophages by 3-hydroxyanthranilic acid, a tryptophan metabolite",
Arch Biochem Biophys, vol. 340, no. 1, 1997 Apr 1 (pp. 117-23)
(3-hydroxyanthranilic acid but not anthranilic acid).
Nordihydroguaiaretic acid and AA861: Lee et al., "Inhibition of
5-lipoxygenase blocks IL-1 beta-induced vascular adhesion
molecule-1 gene expression in human endothelial cells", J Immunol,
vol. 158, no. 7, 1997 Apr 1 (pp. 3401-7). Prostaglandin A1: Rossi
et al., "Inhibition of nuclear factor kappa B by prostaglandin A1:
an effect associated with heat shock transcription factor
activation", Proc Natl Acad Sci USA, vol. 94, no. 2, 1997 Jan 21
(pp. 746-50).
[0046] Sialyl Lewis X (SLe.sup.x) mediates binding of neutrophils
to vascular endothelial cells by binding to E-selectin. (M.
Phillips, et al., Science 1990, 250, 1130; J. Lowe, et al., Cell
1990, 63, 475; T. Feizi, Trends Biochem Sci 1991, 16, 84; M.
Tiemeyer., et al., Proc. Natl. Acad. Sci. USA 1991, 88, 1138; L.
Lasky, Science 1992, 258, 964; and T. Springer, L. A. Lasky, Nature
1991, 349, 196.) Sialyl Lewis X (SLe.sup.x) is a cell surface
carbohydrate ligand found on neutrophils, anchored onto the outer
membrane thereof by integral membrane glycoproteins and/or
glycolipids. Administration of SLe.sup.x inhibits the
SLe.sup.x/E-selectin interaction and blocks adhesion of neutophils
to endothelial cells. (M. Buerke, et al., J. Clin. Invest., 1994,
1140.). Neutrophil-mediated inflammatory diseases may be treated by
administration of Sialyl Lewis X (SLe.sup.x). Selectin inhibitor
include those in the following references. E-, P-, and L-selectin
inhibitors in U.S. Pat. No. 5,830,871. Sulfatides and sialylated or
sulfated fucooligosaccharides, as described in U.S. Pat. No.
5,985,852, and other fucose derivatives as described in U.S. Pat.
No. 5,962,422 and U.S. Pat. No. 5,919,769; as well as described by
Ikami et al., "Synthetic studies on selectin ligands-inhibitors:
Synthesis and inhibitory activity of 2-O-fucosyl sulfatides
containing 2-branched fatty alkyl residues in place of ceramide",
Journal of Carbohydrate Chemistry, vol. 17, no. 3, 1998 (pp.
453470) (sulfated 2-O-alpha-L-fucopyranosyl
beta-D-galactopyranosides containing 2-branched fatty-alkyl
residues in place of ceramide); Todderud et al., "BMS-190394, a
selectin inhibitor, prevents rat cutaneous inflammatory reactions",
J Pharmacol Exp Ther, vol. 282, no. 3, 1997 Sep (pp. 1298-304)
(selectin antagonist BMS-190394, a structural analog of sulfatide).
TBC-1269 (available from Texas Biotechnology Corp., Houston, Tex.)
and other mannose derivatives: for example, Dupre et al.,
"Glycomimetic selectin inhibitors:
(alpha-D-mannopyranosyloxy)-methylbiphenyls", Bioorganic &
Medicinal Chemistry Letters, vol. 6, no. 5, 1996 (pp. 569-572); Lin
et al., "Synthesis of sialyl Lewis x mimetics as selectin
inhibitors by enzymatic aldol condensation reactions", Bioorg Med
Chem, vol. 7, no. 3, 1999 Mar (pp. 425-33) (D-mannosyl
phosphate/phosphonate derivatives enzymatically prepared as sialyl
Lewis x tetrasaccharide mimics); Kogan et al., "Rational design and
synthesis of small molecule, non-oligosaccharide selectin
inhibitors: (alpha-D-mannopyranosyloxy)biphenyl-substituted
carboxylic acids", J Med Chem, vol. 38, no. 26, 1995 Dec 22 (pp.
4976-84). Leumedins: Endemann et al., "Novel anti-inflammatory
compounds induce shedding of L-selectin and block primary capture
of neutrophils under flow conditions", J Immunol 1997 May 15;
158(10):4879-85 (leumedins are small molecules that inhibit
neutrophil movement into inflamed tissues). Di- and tri-valent
small molecules, mainly 3-carboxyaralkyl-substituted
2-.alpha.-D-mannopyranosyloxy-phenyl unsubstitued, oxygen-, or
nitrogen-substituted alkanes (e.g., oxobutane, piperidine), as
described in U.S. Pat. No. 5,919,768. GSC-150: Wada et al., uEffect
of GSC-150, a new synthetic selectin inhibitor, on skin
inflammation in mice", Japanese Journal of Pharmacology, vol. 71,
no. Suppl. 1, 1996 (Page 302P). Sialyl Lewis x analogs: Kiso et
al., "Studies of selectin binding inhibitors: Synthesis of
sialyl-Lewis x and sialyl-Lewis a epitope analogs containing
2-acetamido derivative of N-methyl-1-deoxynojirimycin", Journal of
Carbohydrate Chemistry, vol. 15, no. 1, 1996 (pp. 1-14) (synthesis
of sialyl-Lewis x (15) and sialyi-Lewis a (17) epitope analogs
containing the 2-acetamido derivative of
N-methyl-1-deoxynojirimycin). Glycolipid sulfatide: Nair et al.,
"Inhibition of immune complex-induced inflammation by a small
molecular weight selectin antagonist", Mediators of Inflammation,
vol. 3, no. 6, 1994 (pp. 459-463). Triterpene glucosides such as
glycyrrhizin: Rao et al., "Glycyrrhetinic acid glycosides are
sialyl Lewis X mimics, and function as selectin inhibitors",
Molecular Biology of the Cell, vol. 5, no. Suppl., 1994 (pp. 480A);
Narasinga et al., "Sialyl Lewis X Mimics Derived from a
Pharmacophore Search Are Selectin Inhibitors with Anti-inflammatory
Activity", Journal of Biological Chemistry, vol. 269, no. 31, 1994
(pp. 19663-19666) (glycyrrhizin, an L-fucose derivative, and a
C-fucoside derivative; Subramanian et al., "Attenuation of renal
ischemia-reperfusion injury with selectin inhibition in a rabbit
model", Am J Surg, vol. 178, no. 6,1999 Dec (pp. 573-6). GM-1925:
Cornell and Bowyer, "Attenuation of lung injury in a rabbit acid
aspiration model using GM-1925, a novel selectin inhibitor",
Surgical Forum, vol. 45, 1994 (pp. 107-110). Diisopropyl
fluorophosphate: Palecanda et al., "Complete inhibition of
cross-linking and activation induced shedding of I selectin by the
serine protease inhibitor diisopropyl fluorophosphate DPF", J
Immunol, vol. 150, no. 8 Part 2, 1993 (page 304A). BR 44-09 and BR
44-096837: Heavner et al., "Multiple binding site involvement in
neutrophil selectin adhesion implications for design of peptide and
carbohydrate inhibitors BIO BR 44-09 BR 44-096840", J Cell Biochem
Suppl, no. 17 Part A, 1993 (p. 342); Dalton et al., Inhibition of
selectin mediated adhesion in-vivo and in-vitro BIO BR 44-09 BR
44-096837", J Cell Biochem Suppl, no. 17 Part A, 1993 (p. 342).
GMP-140: May et al., "GMP-140 P Selectin inhibits human neutrophil
activation by lipopolysaccharide analysis by proton magnetic
resonance spectroscopy BIO BA 93-00 BA 93-130631", Biochem Biophys
Res Commun, vol. 183, no. 3, 1992 (pp. 1062-1069).
Tetrasaccharides: Ushakova et al., "Inhibitory activity of
monomeric and polymeric selectin ligands", Vopr Med Khim, vol. 45,
no. 5, 1999 Sep-Oct (pp. 375-83) (tetrasaccharides SiaLex, SiaLea,
HSO.sub.3Lex, their conjugates with polyacrylamide (40 kDa), and
several other monomeric and polymeric substances; all monomeric
inhibitors were about two orders of magnitude weaker;
PAA-conjugates, containing as a ligand tyrosine-o-sulfate in
addition to one of the above mentioned oligosaccharides, were the
most potent synthetic blockers compared with fucoidan, bi-ligand
glycoconjugate HSO3Lea-PM-sTyr); Bertozzi et al., "Sulfated
disaccharide inhibitors of L-selectin: deriving structural leads
from a physiological selectin ligand", Biochemistry, vol. 34, no.
44, 1995 Nov 7 (pp. 14271-8) (generated a simple small molecule
(lactose 6',6-disulfate) with greater inhibitory potency for
L-selectin than sialyl Lewis x). Panosialins: Shinoda et al.,
"Panosialins, inhibitors of an alpha1,3-fucosyltransferase
Fuc-TVII, suppress the expression of selectin ligands on U937
cells", Glycoconj J, vol. 15, no. 11, 1998 Nov (pp. 1079-83).
CY-1503: Schmid et al., "Carbohydrate selectin inhibitor CY-1503
reduces neutrophil migration and reperfusion injury in canine
pulmonary allografts", J Heart Lung Transplant, vol. 16, no.10,1997
Oct (pp. 1054-61).
[0047] Inhibitors of TLRs (toll-like receptors) and/or other
receptors that are sensitive to the LPS-like compounds associated
with acne lesions can be used to ameliorate the signalling that
induces the cytokines TNF.alpha., IL-1.beta., IL-8, and IL-10, as
shown in FIGS. 6 and 8B, and any other related cytokines that are
induced by the P. acnes bacteria. Diglucosamine-based LPS
antagonists include E5564 and E5531, described by E. Lien et al.,
J. Biol. Chem. 276(3): 1873-80 (2001), and by T. K. Means et al.,
J. Immunol., 166(6): 4074-82 (2001), inhibit certain TLRs.
[0048] Generally, molecules having a molecular weight of less than
about 600 will pass through the skin, and lipophilic molecules are
preferred (or a conjugate having a lipophilic portion).
Accordingly, while short chain peptides are not listed above, those
having a low molecular weight and a high proportion of lipophilic
amino acid residues are likely to be useful as topical inhibitors
of AP-1, NF-.kappa.B, elastase, and/or selectin.
[0049] FIGS. 7A-7F show the effect of a control and some of these
inhibitors on acne-affected skin. Each of these figures is an
in-situ zymogram showing collagenase (MMP-1 and/or MMP-8) activity
in a biopsied section; green is fluorescently-labelled collagen
placed on a slide, over which is placed a biopsy section from an
acne lesion from a human volunteer. FIG. 7A is a zymogram of a
biopsy of uninvolved (not acne-affected) skin; there is almost no
collagenase activity. FIG. 7B is a zymogram of acne-involved skin;
there is significant collagenase activity as evidenced by the dark
(black) areas where the fluorescently-labelled collagen has been
degraded by the collagenase in the biopsied specimen. FIG. 7C is a
zymogram of acne-involved skin which was treated with a control
compound C1006 structurally analogous to known inhibitors but found
to be inactive (the subject compound is applied over the biopsy
section laid on the fluorescently-labelled collagen-coated slide);
as seen by the dark areas, there was still significant collagen
degradation (and hence collagenase activity). FIG. 7D is a zymogram
of acne-affected skin treated with a collagenase inhibitor AG 3340
(Drugs R D 1999 February; 1 (2): 137-8); the amount of collagenase
activity is minimal and comparable with that seen for uninvolved
skin. FIG. 7E is also a zymogram of acne-involved skin treated with
collagenase inhibitor GM1489; again there is significant inhibition
of collagenase activity. Finally, FIG. 7F is a zymogram of
acne-involved skin treated with GM6001; again there is significant
suppression of collagenase activity. The MMP inhibitor GM6001 is
N-[(2R)-2-hydroxamidocarbonylmethyl).sub.4-methylpentanoyl]-L-tryptophan
methylamide (ilomastat) (see R E Galardy et al., Ann. NY Acad.
Sci., 732:315-323 (1994)). The inhibitor GM1489 is
N-[(2R)-2-(carboxymethyl)-4-methylpentanoyl]-L-tryptophan
methylbenzylamide (see W M Holleran et al., (1997) Arch. Dermatol.
Res. 289:138-144). The control compound C1006 is
N-t-Butyloxycarbonyl-L-leucyl-L-tryptophan methylamide. These three
compounds (GM6001, GM1489, and C1006, were obtained from AMS
Scientific Inc., Concord, California).
[0050] The compositions of this invention can be provided in any
cosmetically suitable form, preferably as a lotion or cream, but
also in an ointment or oil base, as well as a sprayable liquid form
(e.g., a spray that includes the MMP inhibitor in a base, vehicle,
or carrier that dries in a cosmetically acceptable way without the
greasy appearance that a lotion or ointment would have if applied
to the skin).
[0051] In addition, the compositions contemplated by this invention
can include one or more compatible cosmetically acceptable
adjuvants commonly used, such as colorants, fragrances, emollients,
humectants, and the like, as well as botanicals such as aloe,
chamolile, and the like.
[0052] When used topically, an inhibitor (of a dermal
matrix-degrading enzyme) is used preferably at concentrations of
between about 0.05% and about 5%, more preferably between 0.1% and
1%; antioxidants are preferably taken in "megadoses" (e.g., at
least 1 g/d of vitamin C, at least 1000 I.U. of one or more
tocopherols). A direct inhibitor includes AG3340, used at
0.3%.+-.0.1%.
[0053] In view of the foregoing, another facet of this invention is
the use of an MMP inhibitor in combination with a clinical therapy
for acne. The various treatments for acne, as noted in the
Background section, involve the topical or oral administration of
any number of active ingredients, ranging from antibacterials to
anti-inflammatories. By virtue of this invention, combination
therapies, such as combined oral and topical administration of
tetracycline (which may involve use of two different
tetracyclines), combined oral and topical administration of a
retinoid, or a combination topical composition containing (i) an
MMP inhibitor and/or an elastase inhibitor and (ii) another
compound (such as an antibiotic, comedolytic, and/or
anti-inflammatory).
[0054] In another aspect this invention includes an improved
process for treating acne. As mentioned above, retinoids are known
and presently used for treating acne. According to this invention,
the improvement to that process is the use of a compound that
inhibits the degradation of the retinoid. One enzyme that degrades
retinoids and can be inhibited is cytochrome P-450. In the skin,
retinoids are converted into retinoic acid (RA) as the active form.
Natural retinoids that function in the skin are all trans or are
metabolized to all trans. Retinoic acid (RA; all trans) is
metabolized to inactivation by hydroxylation (via RA 4-hydroxylase)
to 4-hydroxy-RA, which is then oxidized by a reaction mediated by
the cytochrome P450-dependent monooxygenase system. (S. Kang et
al., "Liarczole Inhibits Human Epidermal Retinoic Acid
4-Hydroxylase Activity and Differentially Augments Human Skin
Responses to Retinoic Acid and Retinol In Vivo," J. Invest.
Dermatol., 107:183-187 (August 1996); E. A. Duell et al., "Human
Skin Levels of Retinoic Acid and Cytochrome P-450-derived
4-Hydroxyretinoic Acid after Topical Application of Retinoic Acid
In Vivo Compared to Concentrations Required to Stimulate Retinoic
Acid Receptor-mediated Transcription In Vitro," J. Clin. Invest.,
Skin Retinoid Levels and Reporter Gene Activity, 90:1269-1274 (Oct.
1992); E. A. Deull et al., "Retinoic Acid Isomers Applied to Human
Skin in Vivo Each Induce a 4-Hydroxylase That Inactivates Only
Trans Retinoic Acid," J. Invest. Dermatol., 106:316-320 (February
1996); the disclosures of which are incorporated herein by
reference). Accordingly, compounds which interfere with the
elimination metabolism of all trans RA, the active metabolite of
topically applied retinoids such as 9-cis RA and 13-cis RA, will
beneficially increase the amount of RA in the skin. Thus,
preventing the degradation of natural (all trans) RA in the skin
effectively increases its concentration, and so provides the
benefits useful for its treatment of acne.
[0055] Retinoids that are or may likely be useful for treating acne
include natural and synthetic analogs of vitamin A (retinol),
vitamin A aldehyde (retinal), vitamin A acid (retinoic acid (RA)),
including all-trans, 9-cis, and 13-cis retinoic acid), etretinate,
and others as described in EP-A2-0 379367, U.S. Pat. No. 4,887,805,
and U.S. Pat. No. 4,888,342 (the disclosures of which are all
incorporated herein by reference), and the dissociating retinoids
that are specific for AP-1 antagonism (such as those described by
Fanjul, et al. in Nature (1994) 372:104-110). Various synthetic
retinoids and compounds having retinoid activity are expected to be
useful in this invention, to the extent that s they exhibit
anti-MMP activity in vivo, and such are described in various
patents assigned on their face to Allergan Inc., such as in the
following U.S. Patents, numbered: U.S. Pat. Nos. 5,514,825;
5,698,700; 5,696,162; 5,688,957; 5,677,451; 5,677,323; 5,677,320;
5,675,033; 5,675,024; 5,672,710; 5,688,175; 5,663,367; 5,663,357;
5,663,347; 5,648,514; 5,648,503; 5,618,943; 5,618,931; 5,618,836;
5,605,915; 5,602,130. Still other compounds described as having
retinoid activity are described in other U.S. Patents, numbered:
U.S. Pat. Nos. 5,648,563; 5,648,385; 5,618,839; 5,559,248;
5,616,712; 5,616,597; 5,602,135; 5,599,819; 5,556,996; 5,534,516;
5,516,904; 5,498,755; 5,470,999; 5,468,879; 5,455,265; 5,451,605;
5,343,173; 5,426,118; 5,414,007; 5,407,937; 5,399,586; 5,399,561;
5,391,753; and the like, the disclosures of all of the foregoing
and following patents and literature references hereby incorporated
herein by reference.
[0056] Examples of compounds dermatologically acceptable and having
or likely to have inhibitory effects on the P-450-mediated
degradation of RA and other retinoids include azoles, especially
triazoles, including, for example, ketoconazole (U.S. Pat. Nos.
4,144,346 and 4,223,036), fluconazole (U.S. Pat. No. 4,404,216),
itraconazole (U.S. Pat. No. 4,267,179), liarozole, irtemazole, and
the like; compounds related to these that may also be useful
include, for example, diazines such as flucytosine.
[0057] It would also be beneficial to use such cytochrome P-450
inhibitors in combination with a reduced amount of retinoid; the
P-450 inhibitor decreases the metabolic elimination of the retinoid
and so less retinoid is needed to achieve the same result. Still
further, analytical methods are available for determining whether a
given compound inhibits the degradation of RA by applying the
compound and testing for changes in CRABP (cytoplasmic retinoic
acid binding protein), which will have increased levels if the
levels of RA are also increased by the topical application of the
test compound.
Methods Used in the Examples
[0058] The references noted in this section are incorporated herein
by reference.
[0059] Preparation of skin supernatants for biochemical analysis.
Skin samples were ground by mortar and pestle under liquid
nitrogen, and homogenized in a Dounce tissue grinder in buffer
containing 10 mM Hepes, 1 mM EDTA, 5 mM EGTA, 10 mM MgCl.sub.2, 50
mM glycerophosphate, 5 mM NaVO.sub.4, 2 mM DTT, 0.5 mM PMSF, 10
.mu.g/ml aprotinin, 10 .mu.g/ml leupeptin, and 10 .mu.g/ml
pepstatin, and 0.5% NP-40. Homogenates were centrifuged at 14,000 g
for 15 min., and supernatants were collected and used for
biochemical determinations as described herein.
[0060] Immunohistology: Immunihistology of Type I pN collagen,
MMP-1, and neutrophil elastasewere performed as has been described
by Griffiths, C. E. M., et al., N. Engl. J. Med., 329:530-535
(1993). Type I pN collagen was detected with mouse monoclonal IgG1
antibody (SP1.D8; available from Univ. of Iowa Dept. of Biological
Sciences Developmental Studies Hybridoma Bank, Iowa City, Iowa)
raised against the aminopropeptide region of human Type I
procollagen (Foellmer, H. G., et al., Euro. J. Biochm., 134:183-189
(1983)). The MMP-1 antibody is available from Comicon (Temecula,
Calif.), and the neutrophil elastase antibody is available from
DAKO (Carpinterina, Calif.).
[0061] Western analysis of proteins. Immunoreactive proteins were
visualized by enhanced chemiluminescence detection and quantified
by laser densitometry, or by enhanced chemifluorescence detection
and quantified by a Storm imager (Molecular Dynamics, Palo Alto,
Calif.).
[0062] In-situ Zymography: performed as described by Fisher et al.
in "Pathophysiology of premature skin aging induced by ultraviolet
radiation," New Engl. J. Med., vol. 337, pp. 1419-1428 (1997).
[0063] The foregoing description is meant to be illustrative and
not limiting. Various changes, modifications, and additions may
become apparent to the skilled artisan upon a perusal of this
specification, and such are meant to be within the scope and spirit
of the invention as defined by the claims.
* * * * *